The ryanodine receptor type 1 (RyR1) plays a critical role in skeletal muscle contraction by releasing the Ca2+ required for excitation-contraction coupling. Missense mutations in this enormous homotetrameric Ca2+ channel can cause debilitating skeletal muscle disorders including malignant hyperthermia (MH). However, the overall structure of the RyR1 macromolecular complex and the effects of these mutations on this structure are largely unknown. The short-term objective of this proposal is to develop an innovative method to map the overall tertiary and quaternary structure of RyR1 using Forester resonance energy transfer (FRET) techniques. This biophysical method provides unique structural information by accurately measuring distances between defined positions in the protein. FRET can then be used to determine how these distances change when the protein changes conformation. The long-term objective of the proposed work is to map both wild type and MH mutated RyR1 using this method and thereby gain novel insights into the structure of this protein. Hypothesis: A FRET-based experimental system comprised of an N-terminally fused GFP donor and a fluorescence acceptor targeted to His tags in the primary sequence of RyR1 can be used to measure intramolecular distances within the RyR structure. Specific Aim: We will establish a FRET-based assay to measure distances between defined primary sequence elements of RyR1 by placing both a FRET donor and an acceptor molecule onto the surface of this protein. This FRET pair will consist of an N-terminally fused green fluorescent protein (GFP;fluorescence donor) and fluorescence acceptors (NTA-1 and NTA-Rho) synthesized by the PI that bind to poly-histidine tags strategically placed into well-defined regions of the RyR1 primary sequence. Point to point distances from the N-terminal GFP to these His tags will be computed from FRET measured either as a decrease in donor fluorescence (for NTA-1) or both donor fluorescence quenching as well as enhancement of acceptor fluorescence (for NTA-Rho). We will conduct both cell-based as well as in vitro FRET measurements of GFP- RyR1 fusion proteins in order to estimate the contribution of FRET signals arising from neighboring RyR1 channels. Perspective: Through this integrated series of experiments, we will develop a new set of molecular tools to site-specifically label RyR1 and then examine the structure of this protein using FRET. These experiments will set the stage for future experiments that have the potential to provide unprecedented glimpses of the structure of RyR1 and how this structure is changed by disease-causing mutations. PUBLIC HEALTH RELEVANCE. The proposal will establish a FRET-based technique to visualize the structure of RyR1. This technique will open the door for further structural measurements of RyR1 to determine how disease-causing mutations alter the structure of the channel.